Urine Proteins
Currently, no CZE methods have been approved for the separation of urine proteins by the FDA. Various authors, however, have developed techniques that have used CZE to separate urine proteins. These reports may well help in the eventual introduction of such methods. A limiting factor appears to be salt concentration of urine as well as degraded protein fragments, which can present a very confusing picture. Fig. 2. C Immunosubtraction patterns of an IgM kappa monoclonal gammopathy. Fig. 2. C...
Introduction Analysis Of Urinary Drugs In Clinical And Forensic Toxicology
Clinical and forensic toxicology is concerned with the detection, identification, and measurement of toxic compounds and their metabolites in human body fluids and tissues. Most often the toxic compounds are drugs taken either accidentally or intentionally in quantities sufficient to cause an adverse reaction or death. Analysis and identification of a possible drug or drug combinations, toxicological drug screening and confirmation should encompass as many different classes of drugs as...
Info Ouo
The investigators listed in Table 1 have all determined that CZE will give results comparable to HRAGE for routine the electrophoretic separation of serum proteins in the clinical laboratory. However, as with any procedure, correlation between methods depends on many factors. As such, normal ranges should be established in each individual laboratory. As a comparison, normal values obtained by CZE vs HRAGE in my laboratory are given Table 2. We found that for the five standard fractions of a...
Cze Implementation
When instituting any new procedure in the clinical laboratory, comparative studies between the old and new methodology must be run. When comparing HRAGE and CZE, one should not expect separation of proteins on instrumentation as different as these two methods to give identical results. Because of the difference of detection methods of HRAGE and CZE dye binding vs absorbance at 214 nm , the inter-individual variations by CZE were less than HRAGE 23 . However, variations in patient populations do...
References 1
1. Janini, G. M., and Issaq, H. J. 1993 The buffer in capillary zone electrophoresis, in Capillary Electrophoresis Technology, Guzman, N.A., ed. , Marcel Dekker, Inc., New York, pp. 119-160. 2. Cifuentes, A. and Poppe, H. 1994 Rectangular capillary electrophoresis some theoretical considerations. Chromatographia 39, 391-404. 3. Virtanen, R. and Kivalo, P. 1969 Quantitative high-voltage zone electrophoresis method. Suomen Kemistilehti B 42, 182. 4. Jorgensen, J. W. and Lukacs, K. D. 1981 Zone...
Precision
The precision of both peak area used for quantification and migration time used for identification in CE is not as good as that in HPLC. However, studies aimed at understanding this problem will improve precision, as has been the case with other emerging techniques. In many instances, imprecision in migration time is related to the interaction of the compound with the capillary wall and to changes in the EOF. Fortunately, in CE the migration time is more predictable than the peak height area ....
Applications For The Clinical Laboratory
The applications discussed in this chapter start with methylmalonic acid, since this is the one with which the authors have the most experience. An assay for urine methylmalonic acid has been operating on a routine basis in the first author's clinical laboratory since 1994. Several other applications share many characteristics with methylmalonic acid, and are discussed next, including succinic acid, oxalic and citric acids, and the simple short-chain organic acids. Most of these assays use...
Transient Isotachophoresis
Isotachophoresis see Chapter 2 for a detail description is a powerful method for concentrating drugs on the capillary. It is well-suited for samples with a high salt content. However, coupling it to CE is difficult. A transient isotachophoretic step or self-stacking occurring in the early stages of the electrophoresis is more practical. Under these conditions a suitable, complementary ion is added to the sample to act as a leading terminating ion 25-27 and a large volume is injected. The...
Serum Bilirubin
MECC in conjunction with LIF can separate and detect four bilirubin species, the unconjugated form, monoester and diester conjugates of bilirubin, and bilirubin covalently linked to albumin, directly from human serum. This method can also detect bilirubin at concentrations much lower than that detected by routine visible light absorption methods 32 . Although it is possible to use CE to separate and detect various forms of bilirubin, it is unlikely that a CE assay will be used in the clinical...
Capillary Isotachophoresis cITP
In this technique the sample plug is introduced between two different buffers. One of these, the leading electrolyte, has the highest mobility in the separation. The second, trailing electrolyte, has a mobility lower than anything else does. The sign of the charge on the analytes and the buffers must be the same. When the voltage is applied the ions in the sample form discrete zones that are not separated into peaks one zone is adjacent to the next. The concentration of the analyte within a...
Introduction Ynx
The development of methods for the amplification and detection of specific regions of the DNA molecule using the polymerase chain reaction PCR has resulted in rapid and dramatic advances in biochemical analysis 1 . With the advent of the PCR it is now possible to easily produce analytically significant amounts of a specified DNA product. A typical PCR reaction can produce microgram quantities of target DNA, allowing rapid and efficient screening of genetic defects, cancer susceptibility, and...
Methods Of Separation
4.1. Micellar Electrokinetic Capillary Chromatography MEKC Pure mixtures have been used in the great majority of the studies involving separation of amino acids. However, low-peak capacity of the methods used limited the use of CE in separating more complex mixtures. Using micellar electrokinetic capillary chromatography MEKC , separation of neutral along with charged components was possible based on their partitioning between the aqueous phase and a pseudo-stationary phase 46 . MEKC was shown...
Microchip Technology
The challenges of microchip technology have also been applied to CE analysis of amino acids. By fusing a cover glass over glass or quartz plates into which small channels were micromachined, tiny capillary channels are formed. Such channels function as open-tubular capillaries on the microchip and was used by Seiler et al. 91 to rapidly separate phenylthio-carbamyl amino acids in lt 2 min. Amino acids, derivatized with o-phthaldialdehyde have been separated within a few seconds using either a...
Serum and Urine Paraprotein Capillary Electrophoresis
Most requests for serum and urine electrophoresis occur because of the reversal of albumin globulin ratios in the general chemistry profile or the presence of protein in the urine. The suspicion of possible myelomatosis by the physician also alerts him her to rule out the presence of a monoclonal gammopathy in the serum or light chain in the urine. Because of the increased sensitivity afforded by the newer electrophoresis systems, paraproteins are found more often. Their appearance in a number...
Contributors
Bode Adesoji Department of Pathology, University of Texas Medical Branch, Galveston, TX Nebojsa Avdalovic Dionex Corporation, Sunnyvale, CA John M. Butler GeneTrace Systems Inc., Alameda, CA Jitka Caslavska Department of Clinical Pharmacology, University of Bern, Bern, Switzerland Samir Cherkaoui Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, Switzerland Randall D. Craver Department of Pediatrics, Louisiana State University School of Medicine, New Orleans, LA Mauro...
References Ppd
1. Kyle, R. A. 1993 Benign monoclonal gammopathy after 20-35 years of follow up. Mayo Clin. Proc. 68, 26-36. 2. Chang, C.-Y., Fritsche, H. A., Classman, A. B., Mc Clure, K. C., and Liu, F. J. 1997 Underestimation of monoclonal proteins by agarose gel serum protein electrophoresis. Ann. Clin. Lab. Sci. 27, 123-129. 3. Jolliff, C. R. and Blessum, C. R. 1997 Comparison of serum protein electro-phoresis by agarose gel and capillary zone electrophoresis in a clinical setting. Electrophoresis 18,...
Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia CAH also known as the adrenogenital syndrome can be considered as a family of inborn error of steroidogensis see Fig. 1 . All CAH variants are inherited as autosomal recessive traits. Each member of this family is characterized by a specific enzyme deficiency that impacts cortisol production by the adrenal cortex, and if severe enough can lead to sexual ambiguity in both males and females. The enzymes usually affected are 21-hydroxylase types I and II , beta...
Conclusion Nak
CZE offers an alternative way of evaluating serum samples for paraproteinemias. The two automated instruments available and approved by the FDA offer a time saving and labor saving approach to these assays. There are a number of other instruments that have greater flexibility with regard to various parameters such as temperature, time of separation, changes in buffer, and capillary size. However, these instruments are considered research platforms and as such can offer a great number of...
Conclusion Rsx
The use of CE in combination with immunoassay opens up some interesting avenues of investigation, specifically in the ability simultaneously to screen and detect multiple analytes. The development of CE-based multianalyte immunoassays is limited only by the ability to separate the analytes from each other and from the Ab-Ag complex. As with other CE methods, the multianalyte CE-based immunoassay is rapid, sensitive if using LIF , and reliable for screening purposes. In addition, custom-made...
Info Chc
Fig. 1. CE-LIF competitive immunoassay of four drugs in urine. This figure refers to the separation of four drugs in urine. A fused silica capillary 27 cm x 20 was used for separation. Buffer was 200 mM borate, pH 10.2, with LIF detection 633 mn excitation an 665 nm detection , voltage was 15 kV. The top figure is blank urine and the bottom figure is a urine spiked with the four drugs of interest. Cy5 is a negatively charged fluorescent cyanine dye. The drug concentrations were 73 nM 25 ng mL...
References Stc
1. Jorgenson, J. W. and Lukacs, K. D. 1981 High-resolution separations based on electrophoresis and electroosmosis. J. Chromatogr. 218, 209-216. 2. Jorgenson, J. W. and Lukacs. K. D. 1981 Zone electrophoresis in open-tubular glass capillaries. Clin. Chem. 27, 1551-1553. 3. Causse, E., Terrier, R., Champagne, S., Nertz, M., Valdiguie, P., Salvayre, R., and Couderc, R. 1998 Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection. J....
Quirino J.p. Terabe S. Anal. Chem. 1998 70 1893-1901.
1. Berne, R. M. and Matthew, N. 1988 in Physiology, 3rd ed. Mosby Year Book, St. Louis, MO. 2. Rumsy, G. and Avery, C. J. 1998 Genotype-phenotype analysis in late onset 21-hydroxylase deficiency in comparison to the classical forms. Clin. Endocrinol. 48, 707. 3. Ko, T. M. and Kao, C. H. 1999 Congenital hyperplasia. Molecular characterization. J. Reprod. Med. 43, 379-386. 4. Bose, H. S., Baldwin, S., and Miller, W. L. 1998 Incorrect folding of steroidogenic acute regulatory protein StAR in...
Historical Perspective
In 1937 Tiselius 1 reported on a methodology that was to become one of the most novel ways by which proteins could be separated from one another. His apparatus, although large in size relative to current systems, established the serum protein profile still used today. The apparatus, a U-tube, separated the serum proteins in the liquid phase into five fractions, albumin, a-1, a-2, P, and y globulins. Through the years chemical pathologists and clinical chemists have refined both the methodology...
Physiological Effects Of Glucocortioids
Of the naturally occurring steroids only cortisol, corticosterone, cortisone, and 11-dehydrocorticosterone have appreciable glucocorticoid activity. Cortisol, which is found in the highest concentration, accounts for most of this activity. About 75 of plasma cortisol is bound to cortisol binding globulin CBG, an alpha globulin , 15 is bound to plasma albumin, and 10 is unbound free , representing the physiologically active portion. CBG also has a high binding affinity for progesterone,...
Comparison Of Cze With Hrage
Chapter 4 discussed the classification of serum protein dyscrasias using CZE and HRAGE. This chapter covers paraproteinemia and the comparison of CZE vs HRAGE. CZE appears to offer an alternative to HRAGE for the detection of paraproteinemias in serum. When we used the Paragon CZE 2000, we obtained a 100 concordance between CZE and HRAGE in the detection of monoclonal gammopathies 3 . However this was only when the paraproteins were within the detection limits of HRAGE IgG 0.5 g L, IgA 0.75 g...
Electrophoresis Methods
Multiple methods such as agarose gel 4 , polyacrylamide 8 , isoelectric focusing 8,9 , and two-dimensional 10 electrophoresis have been described to measure the protein profiles associated with the various diseases of the CNS. All of these methods are labor intensive and require an approx 40-fold preconcentration of the CSF in order for the protein bands to be visualized with the stains used e.g., Coomassie brilliant blue . Because of problems associated with preconcentration, a method using...
References Xhs
1. Gofman, J. W., DeLalla, O., and Glazier, F. 1954 The serum lipoprotein transport system in health, metabolic disorders, atherosclerosis and coronary heart disease. Plasma 2, 413-484. 2. Patsch, J. R., Sailer, S., Kostner, G., Sandhofer, F., Holasek, A., and Braunsteiner, H. 1974 Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation. J. Lipid. Res. 15, 356-365. 3. Chung, B. H., Wilkinson, T., Geer, J. C., and Segrest, J. P. 1980 Preparative and...
Adrenal Gland
The adrenal glands are paired structures situated above the kidneys that are approx 2-3 cm wide and 6 cm long and weigh approx 5 g. The glands consists of a yellow, outer cortex that constitutes approx 80 of the adrenal gland and a gray, inner medulla 1 . The adrenal cortex consists of three distinct layers or zones of cells. The outermost layer, the zona glomerulosa, is the site of aldosterone synthesis, the principal mineralocorticoid produced by the human adrenal cortex, and corticosterone...
References Xms
1. Mukherjee, P. S. and Karnes, H. T. 1996 Ultraviolet and fluorescence derivatization reagents for carboxylic acids suitable for high performance liquid chromatography a review. Biomed. Chromatogr. 10, 193-204. 2. DeBacker, B. L. and Nagels, L. J. 1996 Potentiometric detection for capillary electrophoresis determination of organic acids. Anal. Chem. 68, 4441-4445. 3. Lehmann, R., Voelter, W., and Liebich, H. M. 1997 Capillary electrophore-sis in clinical chemistry. J. Chromatogr. B 697, 3-35....
Cebased Immunoassay
CE evolved from the investigation of column electrophoresis by Hjerten 1 and Castimpoolas 2 . Later, Martin and Everaets developed a capillary isotachophoresis system that became commercially available in the mid-1970s. Subsequently, Jorgen and coworkers demonstrated the analytical potential of CE using 75- m fused silica capillaries 3-5 leading to the adaptation and modification of CE for specific applications. The main advantage of CE when used as a separation technique is the small...
Cems
Formic acid Ammonium formate Ammonium acetate Ammonium carbonate Partial-filling technique Electrochromatography MeOH MeCN mixture containing volatile electrolytes the presence of high ionic strength buffers, the opposite required for optimum ESI-MS to produce gas phase ions. The scan rate of MS may not be adequate to reflect the high separation efficiency peak width of a few seconds and resolution of CE. However, by using selected ion monitoring mode SIM on single quadrupole instruments,...
Introduction Ndh
Capillary isoelectric focusing cIEF for analysis of hemoglobin Hb variants has two major clinical applications 1 diagnosis and management of congenital hemoglobinopathies, and 2 assessment of glycemic control in subjects with diabetes. For congenital Hb disorders, like sickle-cell disease or beta thalassemia, the specific identification and precise quantification of both normal and abnormal structural Hb variants is required. Assessment of glycemic control for diabetes management, on the other...
Introduction 1
Protein analysis has become very important to the biotechnology, pharmaceutical, and food industries. Analytical techniques that provide fast, automated analysis, but still give high resolution separation of proteins derived from various sources, such as plasma, blood, and vaccine products, are in high demand. Chromatography and electrophoresis continue to be the preferred means for analyzing proteins. Capillary electrophoresis CE , a micro-format of electrophoresis, has been successfully used...
References Xma
1. Mullis, K. B. and Faloona, F. A. 1987 Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-350. 2. Erlich, H. A. ed. 1989 PCR Technology. Stockton Press, New York, NY. 3. Gibbs, R. A. 1990 DNA Amplification by the polymerase chain reaction. Anal. Chem. 62, 1202-1214. 4. Kasper, T. J., Melera, M., Gozel, P., and Brownlee, R. G. 1988 Separation and detection of DNA by capillary electrophoresis J. Chromatogr. 458, 303-312. 5. Cohen, A. S.,...
Capillary Gel Electrophoresis CGE 221 Mechanism
Capillary gel electrophoresis CGE is separation based on viscous drag. In this mode of CE the capillary is filled with a gel or viscous solution. EOF is often suppressed so that the migration of the analytes is solely by electro-phoresis. Larger molecules tend to be retarded more by the viscous separation medium than are smaller molecules, so that the separation is effectively based on the molecular size. This is the method of choice for molecules that differ in size but not in mass charge...
Au
Fig. 4. Capillary electropherogram of a mixture of apolipoprotein A-I and A-II. Capillary fused silica 50 cm x 50 m running conditions 20 kV buffer BioRad LLV evaluation buffer detection UV at X 220 nm equipment Bio-Rad BioFocus 3000 capillary electrophoresis system. Fig. 4. Capillary electropherogram of a mixture of apolipoprotein A-I and A-II. Capillary fused silica 50 cm x 50 m running conditions 20 kV buffer BioRad LLV evaluation buffer detection UV at X 220 nm equipment Bio-Rad BioFocus...
References Cim
1. Hjerten, S. 1967 Free-zone electrophoresis. Chromatogr. Rev. 9, 122-219. 2. Castimpoolas, N. 1971 Scanning density gradient isoelectric separation of proteins on a mole scale. Sep. Sci. 6, 435. 3. Jorgenson, J. W. and Lukacs, K. D. 1981 Zone electrophoresis in open-tubular glass capillaries. Anal. Chem. 53, 1298. 4. Jorgenson, J. W. and Lukacs, K. D. 1981 High resolution separations based on electrophoresis and electro-osmosis. J. Chromatogr. 218, 209. 5. Jorgenson, J. W. and Lukacs, K. D....
Advantages And Disadvantages Of Cze
For the separation of paraproteins in serum, CZE is comparable to HRAGE. The separations obtained by CE in the beta globulin region where transferrin can potentially interfere with IgA MC proteins appear to be superior to HRAGE. This also appears to be true for IgM MC proteins. No application artifacts are seen with the CZE procedure and the concentrations of the proteins in the individual peaks are not dependent on staining differences for the individual proteins. This is particularly...
Info Kvt
Fig. 1. Separation of basic proteins using a polymer coated capillary. Conditions 50-cm total length 45 cm to detector 50 m i.d. Electrolyte 50 mM sodium acetate, pH 4.5 20 kV 400 V cm gravity injection 50 mm x 10 s detection UV 210 nm sample concentration, 100 g mL. Peak identification 1 lysozyme chicken egg white 2 cytochrome c bovine heart 3 ribonuclease A bovine pancreas , 4 myoglobin horse skeletal muscle and 5 a-chymotrypsinogen A bovine pancreas . ing extremes of pH, because this can...
Info Hxu
55. Neubeck, W., Wieland, H., Habenicht, A., Muller, P., Baggio, G., and Seidel, D. 1977 Improved assessment of plasma lipoprotein patterns. III. Direct measurement of lipoproteins after gel-electrophoresis. Clin. Chem. 23, 1296-1300. 56. Everaerts, F. M., Beckers, J. L., and Verheggen, T. P. E. M. 1976 Isotachophoresis Theory, Instrumentation and Applications, Amsterdam, The Netherlands, Elsevier, pp. 1-384. 57. Bocek, P., Gebauer, P., Dolnik, V., and Foret, F. 1985 Recent developments in...
Cze Instrumentation
CZE instrumentation offers a semi-automated Bio-Focus 2000 or completely automated Paragon CZE 2000 system. These instruments elimi- Fig. 4. Dysproteinemias as identified by CE. Fig. 4. Dysproteinemias as identified by CE. nate many of the errors mentioned in the previous section that are associated with conventional HRAGE methods. Labor costs are also decreased with CZE since there is no time spent in specimen application, fixing, staining, washing, and drying of the gels. Both methods,...
Conclusion 1
The CZE procedure offers many advantages in labor saving, cost per test, and reproducibility compared to the HRAGE procedure. The literature on this procedure, in which comparative studies with HRGE have been reported, have been extremely favorable for its adaptation in the clinical laboratory for serum protein separation. Although it may make sense to replace HRAGE with CZE, the laboratory should not totally discard HRAGE because it will be useful as a backup confirmation method when patterns...
Immunofixation Electrophoresis Vs Immunosubtraction
Immunofixation electrophoresis IFE is in all probability the gold standard for the detection of monoclonal gammopathies in the clinical laboratory and to identify the type as well as the heavy- and light-chain class. It has also replaced the classical immunoelectrophoresis because of its simplicity of interpretation. In addition, it is also an extremely sensitive procedure because of the combination of two proteins, antigen and antibody, which increases the amount of dye uptake almost 10-fold....
Conclusions
CE has made significant progress in the field of bioseparations. With the recent advances in coating chemistries and capillaries the technique has become more robust, particularly for protein analysis. The benefit of improved separation efficiencies, recovery, and reproducibility of protein separations using coated capillaries translates into wider acceptability of CE. As the demand for recombinant drugs increases, the need for analytical techniques that give complementary results increases and...
Cost
Because CE is a separation technique, it will compete with more traditional chromatographic techniques such as gas chromatography GC and HPLC. However, unlike the classical chromatographic techniques, CE can also compete with classical electrophoresis, such as agarose gel and isoelectric focusing. Table 1 gives a typical cost comparison for the procedures that CE could replace in a clinical laboratory. As shown in this table, in certain instances CE can be less expensive. The main cost savings...